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Esti Enjelina
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Sekip Utara, PO BOX BLS 21, Yogyakarta 55281
Chemical Engineering, Chemistry & Bioengineering Chemistry

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Validation of a Non-Specific Dye Real-Time PCR Assay for Porcine Adulteration in Meatball Using ND5 Primer Tri Joko Raharjo; Ery Nourika Alfiraza; Esti Enjelina; Deni Pranowo
Indonesian Journal of Chemistry Vol 17, No 2 (2017)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (359.999 KB) | DOI: 10.22146/ijc.22646

Abstract

Porcine adulteration in meatball samples were analyzed using real-time polymerase chain reaction (RT-PCR), based on the ND5 primer obtained by previous study. This work consisted of three stages which were annealing temperature optimization, method validation, and application. DNA template was extracted using phenol-CIAA (chloroform-iso amyl alcohol) method. The optimum annealing temperature for ND5 primers (forward primer 5'-CATTCGCCTCACTCACATTAACC-3' and reverse primer 5'-AAGAGAGAGTTCTACGGTCTGTAG-3') was 58.0 °C, obtained after testing annealing at 50.5 to 59.5 °C gradient temperature with 5 °C interval. Melting curve analysis was done at 65.0 to 95.0 °C, with increasing temperature for 0.5 °C per 2 sec. Method was validated for its specificity, precision and limit of detection. RT-PCR method with ND5 primers produced 227 bp DNA fragment with 78.50 °C Tm value. From eight commercial meatball samples, one was detected containing porcine. The methods showed high specificity and precision, with experimentally determined limits for porcine were no less than 1%.
Co-Authors Alfiraza, Ery Nourika Deni Pranowo Tri Joko Raharjo